Preventives/remedies for autoimmune demyelinating diseases

ABSTRACT

The present invention is drawn to a method for treating autoimmune demyelinating diseases by administering to a patient in need thereof an effective amount of a Fas antagonist. The method of the present invention uses, for example, as a Fas antagonist a substance which suppresses Fas-Fas ligand binding.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP99/02818 which has an Internationalfiling date of May 28, 1999, which designated the United States ofAmerica.

TECHNICAL FIELD

This invention relates to preventives and remedies for autoimmunedemyelinating diseases which contain an apoptosis-suppressing substanceas their effective component.

BACKGROUND ART

Fas is a cell surface protein which transmits apoptosis signal to thecell, and Fas is recognized by Fas antibody (Yonehara, S. et al., J.Exp. Med., vol. 169, 1747-1756, 1989) which is a monoclonal antibodyproduced by immunizing a mouse with human fibroblast. Fas gene wascloned by Itoh, N. et al., and it was then found out that Fas is a cellmembrane protein of about 45 kD, and from the amino acid sequence, itwas revealed that Fas is a member of TNF receptor family (Cell, vol. 66,pages 233-243, 1991). Mouse Fas gene was also cloned , and theexpression of Fas mRNA in thymus, liver, lung, heart, and ovary wasconfirmed (Watanabe-Fukunaga, R. et al., J. Immunol., vol. 148, pages1274-1279, 1992).

Human Fas ligand is a polypeptide which has been reported by Nagata etal. to be a native molecule which induces apoptosis of Fas-expressingcells (Takahashi, T. et al., International Immunology, vol. 6, pages1567-1574, 1994). Human Fas ligand is a glycosilated type II membraneprotein of TNF family with a molecular weight of about 40 kD. As in thecase of TNF, human Fas ligand in the human body is estimated to be inthe form of a trimer (Tanaka, M. et. al., EMBO Journal, vol. 14, pages1129-1135, 1995). The extracellular domain of the human Fas ligand ishighly homologous with the extracellular domain of rat Fas ligand (Suda,T. et al., Cell, vol. 75, pages 1169-1178, 1993) and mouse Fas ligand(Takahashi, T. et al., Cell, vol. 76, pages 969-976, 1994). The humanFas ligand recognizes not only the human Fas but also the mouse Fas toinduce the apoptosis, and vice versa, the rat Fas ligand and the mouseFas ligand also recognize the human Fas to induce the apoptosis.

Considerable researches have also been done on the mechanism of signaltransduction in the cell upon the Fas-mediated apoptosis, andidentification and cloning of the factor which interacts with theintracellular domain of the Fas, in particular, the region called “deathdomain” to transmit or block the signal have been reported. Possibilityof the involvement of interleukin-1-converting enzyme (ICE)-relatedthiol proteases in the signal transduction of the Fas-mediated apoptosishas also been indicated.

Relationship of the apoptosis, in particular, the Fas-mediated apoptosiswith various diseases and physiological phenomena has been recentlyindicated. For example, possibility has been indicated for involvementof abnormal Fas-mediated apoptosis in the death of hepatocytes in viralfulminant hepatitis, in some types of autoimmune diseases, and the like.

Possibility of the involvement of the Fas/Fas ligand system in functionsother than the apoptosis, for example, in the function of inducinginflammation by acting on neutrophil has also been indicated (Kayagaki,N. et al., Rinshou Meneki (Clinical Immunology), vol. 28, pages 667-675,1996).

Autoimmune diseases are diseases induced by the attack of autoreactivelymphocytes after responding to an autoantigen, and the disease isassociated with various symptoms. The body does not exhibit anyexcessive immunoreaction when normal, and self-tolerance is the stateestablished in a normal body. However, abnormality in immunomodulationmechanism invites production of antibodies against various componentsconstituting the self and emergence of autoreactive lymphocytes. Theautoreactive T cell are normally removed in thymus by apoptosis.However, when such autoreactive T cell transfers to periphery withoutbeing removed in thymus by some abnormality, they are accumulated in theperiphery. Tolerance is also established for B cells, and autoreactive Bcells are normally removed by apoptosis. However, when the autoreactiveB cells fail to be removed by some abnormality, they are alsoaccumulated in the periphery as in the case of the T cells. Theautoimmune disease are caused by such autoreactive lymphocytes.

Autoimmune demyelinating disease is induced by an autoantibody specificto nerve system, and the disease is associated with selectivedestruction of the myelin and the cells constituting the myelin.Histologically, the disease is associated with disappearance of myelin,cellular infiltration in the region surrounding the vein. The disease isassociated with clinical conditions of loss of sight, paresthesia,quadriplegia, and other neurologic manifestation.

Detailed etiology of the demyelinating disease is not fully found out.For example, for the demyelinating inflammation undergoing recurringremission and relapse such as multiple sclerosis include, possibility ofthe involvement of viral infection (Carp, R. I. et al., Prog. Med.Viol., vol. 24, pages 158-177, 1978) is indicated in addition to theinvolvement of autoimmunity (De Keyser, J., Neurology, vol. 38, pages371-374, 1988).

Typical treatments which has been employed for the demyelinating diseaseinclude treatment by nonspecific immunosuppression by the use of animmunosuppressant in combination with ACTH (adrenocorticotropic hormone)(Saida K. Saishin-Igaku (Current Medicine), vol. 10, pages 1963-1971,1991). This treatment, however, failed to exhibit prolonged effects, andmoreover, this treatment is not effective for the disease of chronic,progressive type (Weiner et al., Neurology, vol. 39, pages 1143-1149,1989). In view of such situation various treatments are currentlyinvestigated for specifically suppressing the activity of the autoimmuneT cells, including the administration of T cell vaccine (Ben-Nun, A etal., Nature, vol. 292, pages 60-61, 1981) or T cell receptor vaccine (J.Immunol, vol. 152, page 2510, 1994; J. Immunol, vol. 152, page 2520,1994), oral immunity tolerance (Science, vol. 259, page 1321, 1993),peptide analog (Immunol. Today, vol. 14, page 602-609, 1993), andadministration of anti-CD4 antibody. No results with significanteffectivity has so far been reported except for some of these agent (Tcell receptor vaccination) wherein the effect of reducing the frequencyof autoantigen-reactive T cell in the peripheral blood has beenreported.

When homogenate of spinal cord is subcutaneously inoculated withFreund's complete adjuvant containing killed Mycobacterium tuberculosis,symptom of encephalomyelitis such as paralysis of hind legs is evoked insensitive animals at 10 to 14 days after the inoculation. This is theprototype of the experimental autoimmune encephalomyelitis (EAE). Thisis also a typical model of autoimmune disease derived by immunizing anexperimental animal with a protein antigen or a peptide from brain, andthis model has been extensively investigated from old days lip as adisease model of multiple sclerosis and acute disseminatedencephalomyelitis (ADEM). Analysis of EAE has brought various findingsincluding the involvement of T cell specific to autoantigens such asmyelin basic protein and proteolipid protein expressed in central nervesystem (Ota, K. et al., Nature, vol. 396, pages 183-187, 1990).

Various studies have been recently conducted and reported for therelation between the multiple sclerosis and the apoptosis mediated byFas/Fas ligand system. Sameer, D. et al. reported that they found theFas ligand expressed in microglia cells and infiltrated T cells and theFas expressed in oligodendrocytes, in the lesion of human multiplesclerosis (J. Exp. Med., vol. 184, pages 2361-2370, 1996). Kimberly A.et al. (J. Immunol., vol. 159, pages 3096-3099, 1997) and Hanspeter, W.et al. (J. Immunol., vol. 159, pages 3100-3103, 1997) suggested throughanimal experiment of multiple sclerosis using lpr and gld mouse, whichare genetically deficient of the Fas and the Fas ligand, respectively,that the apoptosis mediated by the Fas/Fas ligand is involved in themultiple sclerosis. In the meanwhile, Eileen, A. et al. (J. Clin.Invest., vol. 98, pages 1602-1612, 1996) and Suzana, M. et al. (J. Exp.Med., vol. 186, pages 507-515, 1997) suggested through animal experimentof the multiple sclerosis using the same lpr and gld mouse that theapoptosis mediated by the Fas/Fas ligand is not involved in the multiplesclerosis. In other words, the relationship between the pathology of themultiple sclerosis and the apoptosis mediated by the Fas/Fas ligandsystem is still unknown and differently conceived depending on theinvestigator. In addition, efficiency of the drug delivery to braintissue is generally low, and it is utterly unknown whether the drugwhich suppresses the apoptosis by the Fas/Fas ligand administered to thebody can suppress the Fas/Fas ligand-mediated apoptosis in the braintissue, and it is also unknown whether the results will be the same asthose obtained in the mouse genetically deficient of the Fas or the Fasligand.

Up until now, there is no preventive and therapeutic agent forautoimmune demyelinating diseases which acts by suppressing theapoptosis, and no therapeutic agent which binds to the Fas ligand hasbeen reported.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a preventive andtherapeutic agent for autoimmune demyelinating diseases which acts bythe novel mechanism of suppressing the apoptosis. More specifically, thepresent invention provides a preventive and therapeutic agent forautoimmune demyelinating diseases which contains anapoptosis-suppressing substance as its effective component and atherapeutic method wherein such agent is used.

The inventors of the present invention have conducted intensive studieson the relation between the apoptosis and the autoimmune demyelinatingdiseases in order to save those suffering from such diseases, and foundthat the pathology is improved in the model of autoimmune demyelinatingdiseases by the apoptosis-suppressing substance. The present inventionhas been completed on the bases of such finding.

Accordingly, the present invention is directed to a preventive andtherapeutic agent as described below.

(1) A preventive and therapeutic agent for autoimmune demyelinatingdiseases containing an apoptosis-suppressing substance as its effectivecomponent.

(2) A preventive and therapeutic agent according to (1) wherein saidapoptosis-suppressing substance is a Fas antagonist.

(3) A preventive and therapeutic agent according to (1) or (2) whereinsaid apoptosis-suppressing substance is a substance which suppressesFas-Fas ligand binding.

(4) A preventive and therapeutic agent according to any one of (1) to(3) wherein said apoptosis-suppressing substance is a Fas derivative.

(5) A preventive and therapeutic agent according to any one of (1) to(3) wherein said apoptosis-suppressing substance is an anti-Fas ligandantibody.

(6) A preventive and therapeutic agent according to any one of (1) to(5) wherein said autoimmune demyelinating disease is a diseaseassociated with demyelinating in central nerve system.

(7) A preventive and therapeutic agent according to any one of (1) to(5) wherein said autoimmune demyelinating disease is at least one memberselected from acute disseminated encephalomyelitis and multiplesclerosis.

(8) A method for preventing and treating autoimmune demyelinatingdiseases wherein an apoptosis-suppressing substance is administered.

(9) Use of an apoptosis-suppressing substance in producing apharmaceutical for preventing and/or treating autoimmune demyelinatingdiseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of the FLIM58 in improving thepathology of the rat EAE model.

FIG. 2 is a graph showing the effect of the FLIM58 in improving the bodyweight loss of the rat EAE model.

REST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in further detail.

The autoimmune demyelinating diseases which are to be treated by thepreventive and therapeutic agent of the present invention includevarious diseases. Such diseases may be roughly categorized into thediseases wherein the demyelination occurs in the central nervous systemand the diseases wherein the demyelination occurs in the peripheralnervous system. Preferably, the diseases wherein the demyelinationoccurs in the central nervous system are treated by the presentinvention.

Typical diseases associated with the demyelination in the centralnervous system are acute disseminated encephalomyelitis and multiplesclerosis and the like. The acute disseminated encephalomyelitis includeidiopathic acute disseminated encephalomyelitis, post infectious acutedisseminated encephalomyelitis, and post vaccinal acute disseminatedencephalomyelitis and the like. Multiple sclerosis includes concentricsclerosis, neuromyelitis optica (Devic's disease), and the like. Thesediseases, and in particular, the multiple sclerosis undergo recurringremission and relapse. Both the disease in the remission phase and therelapse phase are to be treated by the agent of the present inventionacting as a preventive agent and a therapeutic agent, respectively.

The diseases associated with the demyelination in peripheral nervesystem include chronic, inflammatory demyelinating polyradiculitis andacute, inflammatory, demyelinating polyradiculitis and the like. Theacute, inflammatory, demyelinating polyradiculitis includesGuillain-Barre syndrome and the like.

In these diseases, the apoptosis-suppressing substance suppresses theapoptosis occurring in each disease to realize the therapeutic effectsfor the disease. In the remission phase or before the emergence of thepathological condition of the disease, the apoptosis-suppressingsubstance suppresses the apoptosis to realize prophylactic effects forthe disease.

The terms “prevention”, “preventive” and “prophylactic” used in thepresent invention encompass both the prevention of the first occurrenceof the disease and the prevention of the relapse of the disease afterthe remission.

It should be noted that mammals other than human may also be treated bythe agent of the present invention although the human is the mostimportant object of the therapy.

The apoptosis-suppressing substance used in the present invention is notlimited to any particular type as long as it suppresses or inhibits theapoptosis.

Typical apoptosis-suppressing substances are Fas antagonists andsubstances which are capable of suppressing the binding between the Fasand the Fas ligand. The substance employed is not limited to anyparticular type as long as it blocks signal generation by the Fas ortransduction of the thus generated signal at some stage to therebysuppress the function or the biological action of the Fas/Fas ligandsystem (and in particular, the apoptosis). The mechanism of suchblockage may be inhibition of the action, function or expression of theFas ligand or the Fas; interaction with the extracellular domain of theFas ligand or the Fas; inhibition of the Fas ligand-Fas interaction;affecting the interaction between the intracellular domain of the Fasand the intracellular factor which interacts with the intracellulardomain of the Fas; inhibition of the activity of the intracellularfactor (for example, ICE-like protease) which is involved in the signaltransduction of the Fas-mediated apoptosis, and the like. Theapoptosis-suppressing substance may comprise either a high molecularweight protein compound or a low molecular weight compound.

Exemplary apoptosis-suppressing substances include substances which havethe activity of suppressing the Fas-mediated apoptosis, such as a Fasderivative; an anti-Fas antibody; an anti-Fas ligand antibody; anantisense oligonucleotide for the gene of the Fas or the Fas ligand; anantisense oligonucleotide for the mRNA of the Fas or the Fas ligand; asubstance which interacts with the intracellular domain of the Fas; andan ICE inhibitor. The apoptosis-suppressing substances used in thepresent invention is preferably the one which has the function ofsuppressing the Fas-mediated apoptosis such as Fas derivative, anti-Fasantibody, or anti-Fas ligand antibody. Furthermore, the anti-Fasantibody or the anti-Fas ligand antibody is preferably the antibodywhose antigen is the Fas or the Fas ligand from the animal to betreated. For example, the anti-Fas antibody or the anti-Fas ligandantibody used for treating human is preferably the antibody whoseantigen is the Fas or the Fas ligand from human, namely, anti-human Fasantibody or anti-human Fas ligand antibody.

The anti-Fas ligand antibody is preferably a chimeric antibody or ahumanized antibody. An exemplary preferable chimeric antibody which maybe used for treating a human is a chimeric antibody comprising theconstant region from the human antibody and the variable region from anon-human antibody. An exemplary preferable humanized antibody which maybe used for treating a human is a humanized antibody wherein theconstant region and the framework region (FR) are of human origin, andthe complementarity determining region (CDR) is of non-human origin.More preferably, the anti-Fas ligand antibody used in the presentinvention is a reshaped human antibody wherein the CDR from the antibodyof a mammal other than human such as mouse is replaced with the CDR ofthe human antibody. A non-human antibody is associated with biologicaldefects when it is used in treating a human, for example, relativelyshort circulation half life, lack of developing various importantfunctional properties of the immunoglobulin, and immunogenicity.Furthermore, if various mouse monoclonal antibodies or other monoclonalantibodies with the antigenicity against human are developed in futureand one or more such non-human antibodies are used for once or forseveral times, the subsequent administration of such non-human antibodyafter such initial administration may be nullified due to thecrossreactivity even if the subsequent therapy had no relation to theinitial therapy. In such a case, the non-human antibody administeredafter the initial administration may even act as a hazardous substance.The chimeric antibody and the humanized antibody have obviated suchdefects.

The Fas antagonist used in the present invention is preferably the onewhich suppresses the apoptosis of the Fas-expressing cell in anappropriate assay, for example, in the assay described in InternationalPatent Application Publication No. WO 95/13293. This publication citedherein is incorporated herein by reference.

The antibody used in the present invention may be either a polyclonalantibody or a monoclonal antibody, and the molecular species of theantibody used in the present invention is not particularly limited. Theantibody used in the present invention may be either an antibodymolecule of normal form or a fragment thereof as long as the antibodyused is capable of binding to the antigen to inhibit the Fas-mediatedapoptosis. Exemplary antibody fragments include Fab, F(ab′)₂, Fv, andsingle chain Fv (scFv) which is the Fv of heavy chain linked to the Fvof light chain by an adequate linker to form a single chain. Amongthese, an example of the most preferable anti-Fas ligand antibody ismouse F919-9-18 antibody produced by hybridoma F919-9-18 which wasoriginally deposited on Jun. 22, 1995 in National Institute ofBioscience and Human Technology, Agency of Industrial Science andTechnology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan)(Accession No. P-15002) and transferred from the original deposition tothe international deposition on May 9, 1996 (Accession No. FERMBP-5535).

The anti-Fas ligand antibody and the anti-Fas antibody used in thepresent invention may be prepared by known process, for example, by theprocess described in International Patent Application Publication No.WO95/13293 and International Patent Application Publication No.WO97/02290. These publications are herein incorporated by reference.

The chimeric antibody which may be used in the present invention may beproduced by a known chimeric antibody production process. For example, amethod for producing a chimeric protein is described in Example 1 of theInternational Patent Application Publication No. WO 95/13293. Thispublication is herein incorporated by reference.

The humanized antibody used in the present invention may be prepared inaccordance with Riechmann, L. et al. (Nature 332: 323 (1988) andEuropean Patent Publication No. EP-A-0239400); Queen et al. (Proc. Natl.Acad. Sci. USA 86: 10029 (1989), International Patent ApplicationPublication Nos. WO 90/07861 and WO 92/11018); Co et al. (Proc. Natl.Acad. Sci. USA 88: 2869 (1991)); Co et al. (Nature 351: 501 (1991)); Coet al. (J. Immunol. 148: 1149 (1992)), and the like. These publicationsare herein incorporated by reference. A preferable example is humanizedanti-Fas ligand antibody having the CDR derived from the murineF919-9-18 antibody, which is disclosed in International PatentApplication Publication No. WO 97/02290 (Application No.PCT/JP96/01820).

The Fas derivative used in the present invention is not limited to anyparticular type as long as it is capable of binding at least with theFas ligand, or capable of inhibiting the Fas ligand-mediated apoptosis.The Fas derivative may also be the one which comprises an amino acidsequence of a known Fas that has been arbitrarily mutated at one or moreamino acid residues by substitution, deletion or/and addition, and whichinhibits the biological actions of the Fas/Fas ligand system, and inparticular, the Fas-mediated apoptosis, with the binding activity to theFas ligand retained. The Fas derivative may also comprise a mutant ofFas, Fas in a truncated form, a chimeric protein, a fusion protein, anda chemically modified Fas. The Fas from which the Fas derivative isderived may be the one derived from any animal species as long asabove-mentioned property retained, although use of the Fas of humanorigin is preferred in consideration of the antigenicity.

Exemplary Fas derivatives are a known Fas from which the extracellulardomain or the transmembrane domain has been deleted; a chimeric proteinof the extracellular domain of a Fas and another protein such as humanFas-Fc (hFas-Fc) which is a chimeric protein of the extracellular domainof human Fas and Fc fragment of human immunoglobulin. The Fas derivativeis not limited for its production process, and may be the one preparedby utilizing known Fas sequences and known gene engineering techniques.For example, the production process is described in the Example 1 ofInternational Patent Application Publication No. WO 95/13293 andExamples of Application Publication No. WO 97/42319. These publicationsare herein incorporated by reference.

Another preferable Fas derivative is the Fas having a deletion in its Nterminal. Among these, Fas derivatives, the shFas(nd29)-Fc and theshFas(nd29)-hinge (International Patent Application Publication No. WO97/42319) coded in plasmids (pM1304 and pM1317) included in the E. coliwhich were originally deposited in Mar. 14, 1996 in National Instituteof Bioscience and Human Technology, Agency of Industrial Science andTechnology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan)(Accession Nos. P-15514 and P-15515) and transferred from the originaldeposition to the international deposition on Mar. 6, 1997 (AccessionNo. FERM BP-5854 and Accession No. FERM BP-5855) are derivativesincluding the extracellular domain of the known human Fas from which Nterminal sequence of from 1st to 29th amino acid has been deleted, andthese highly active derivatives are preferable examples of the effectivecomponent for the preventive and therapeutic agent of autoimmunedemyelinating diseases of the present invention. This publication isherein incorporated by reference.

The Fas derivatives as described above which may be used in the presentinvention may be confirmed for their activity to bind to the Fas ligandor their activity to suppress the Fas-mediated apoptosis by anappropriate assay method.

The antisense oligonucleotide for the gene of the Fas or the Fas ligandor the antisense oligonucleotide for the mRNA of the Fas or the Fasligand used in the present invention is not limited to any particularsequence as long as it inhibits the expression of the Fas or the Fasligand, and may be, for example, the antisense oligonucleotide of theFas ligand disclosed in Example 20 of International Patent ApplicationPublication No. WO 95/13293. This publication is herein incorporated byreference.

The preventive and therapeutic agent for autoimmune demyelinatingdiseases of the present invention can be used as a therapeutic agent forthe patients suffering from the autoimmune demyelinating diseases, andas a prophylactic agent for the autoimmune demyelinating diseases in thecase of the patients suffering from systemic autoimmune disease, organspecific autoimmune disease except to nerve system, viral infection andthose inoculated with a vaccine and the like diseases. Furthermore, thepreventive and therapeutic agent of the present invention can be used asan agent for preventing relapse of the disease in the case of thepatients suffering from multiple scleroses or other diseases whereinremission and relapse phases are repeated, and who are in the remissionphase.

The preventive and therapeutic agent for autoimmune demyelinatingdiseases of the present invention is characterized by its inclusion ofthe apoptosis-suppressing substance as described above. The agent may bein the form of a pharmaceutical composition or kit wherein the Fasantagonist is appropriately combined with at least one pharmaceuticalcarrier or medium such as sterilized water, physiological saline, avegetable oil, a mineral oil, a higher alcohol, a higher fatty acid, ora nontoxic organic solvent; and optional additives such as an excipient,a colorant, an emulsifier, a suspending agent, a surfactant, asolubilizer, a nonadsorptive, a stabilizer, a preservative, anantioxidative, a buffer, an isotonizing agent, or a pain relievingagent. The agent may be administered either orally, or parenterally byintravenous, intracoronary, subcutaneous, intramuscular, percutaneous,intrarectal, or topical administration or by inhalation.

Preferably, the preventive and therapeutic agent of the presentinvention is parenterally administered by either systemic or topical,rapid or continuous administration.

The preventive and therapeutic agent of the present invention may beadministered to a human at an appropriate dose which may be determinedby taking the conditions and the age of the patient as well as theadministration route into consideration. For example, an adequatedivided dose in the range of approximately 0.01 to 1000 mg/kg may beselected in the case of systemic administration, and within this range,an adequate divided dose in the range of 0.01 to 100 mg/kg may bepreferably selected. The preventive and therapeutic agent for autoimmunedemyelinating diseases of the present invention, however, is not limitedto the administration route and the dose as described above. Two or moreapoptosis-suppressing substance including Fas antagonists, Fas/Fasligand binding-suppressing reagent, and anti-Fas ligand antibody may beused in combination, and in further combination with other drugs.

The preventive and therapeutic agent for autoimmune demyelinatingdiseases of the present invention may be formulated into apharmaceutical preparation in a normal process. For example, aninjection may be prepared by dissolving the purifiedapoptosis-suppressing substance such as the Fas antagonist, the Fas/Fasligand binding-suppressing reagent or the anti-Fas ligand antibody in amedium such as physiological saline or a buffer and optionallysupplementing the solution with an additive such as an anti-adsorptive.The preparation may also be in the form of a lyophilizate which is to bereconstituted before its use, and may contain any of the excipients thatare generally used for facilitating the lyophilization.

The apoptosis-suppressing substance used in the preventive andtherapeutic agent for autoimmune demyelinating diseases of the presentinvention exhibits effects of suppressing organ and tissue disorders inautoimmune demyelinating disease models, in particular, in the model ofdemyelinating disease wherein demyelination occurs in the central nervesystem as shown in the Examples. Although the effects of suppressingorgan and tissue disorders demonstrated in the Examples are theprophylactic and therapeutic effects and synergy thereof, the effects ofpreventing the relapse of the disease can be demonstrated by conductingthe investigation in the relapse model. It should be noted that, in theExamples, an anti-mouse Fas ligand antibody is used in the demonstrationof the therapeutic and prophylactic effects since the models used in theexperiments are mouse models. Equivalent inhibitory effects may beexpected for the anti-human Fas ligand antibody when used in human.

It should be noted that the preventive and therapeutic agent forautoimmune demyelinating diseases of the present invention exhibits notoxicity as demonstrated in the following Examples, and therefore, itcan be used safely. In view of such situation, the preventive andtherapeutic agent for autoimmune demyelinating diseases of the presentinvention is expected to exhibit prophylactic, therapeutic, orameliorating effects for the autoimmune demyelinating diseases.

Next, the present invention is described in further detail by referringto Examples which are given by way of examples and not by way oflimiting the scope of the present invention. The abbreviations used inthe following description are those commonly used in the art.

The production process and the apoptosis-suppressing activity of theanti-Fas ligand antibody, humanized anti-Fas ligand antibody, and theFas derivative of the present invention are disclosed in the Examples ofInternational Patent Application Publication Nos. WO 97/02290 and WO97/42319.

EXAMPLE 1 Production of Anti-mouse Fas Ligand Antibody and itsPurification

(1) Production of Anti-mouse Fas Ligand Antibody

A plasmid containing human elongation factor (EF) promoter, and in itsdownstream, the gene coding for the chimeric protein prepared by fusingthe extracellular Ad) domain of mouse Fas ligand from soluble mouse Fasligand WX2 (J. Immunology, vol. 157, pages 3918-3924, 1996) and theintracellular domain, the transmembrane domain, and a part of theextracellular domain (from N terminal to 78th amino acid) of mouse CD40ligand was prepared (Mizushima-Nagata, Nucleic Acids Research, vol. 18,page 5322, 1990). The plasmid was transfected into the WR19L cell toobtain a recombinant cell W40LFL expressing the mouse Fas ligand on itscell membrane for use as the antigen to be administered. Armenianhamsters were used for the animals to be immunized. The Armenianhamsters were subcutaneously administered with 1×10⁷ W40LFL cells mixedwith Freund's complete adjuvant, and a month later, subcutaneouslyadministered with 2×10⁷ W40LFL cells suspended in PBS, and in another amonth later, administered with 5×10⁶ W40LFL cells suspended in PBS intotheir foot pads. 3 days after the administration, lymph node cells wereisolated and fused with mouse myeloma cell P3-X63-Ag8-U1 (P3-U1). Afterselecting the hybridoma in HAT medium(hypoxanthine-aminopterin-thymidine), hybridoma FLIM58 whose supernatanthad neutralizing activity for cytotoxicity of mouse Fas ligand wasobtained from the survived hybridomas.

(2) Production of FLIM58 and its Purification

Hybridoma FLIM58 was cultured in serum-free medium Hybridoma-SFM (GIBCOBRL), and the culture supernatant was purified by protein A column(PROSEP-A, Bioprocessing) to obtain purified antibody FLIM58.Concentration of the protein was calculated from absorbance at 280 nm.

EXAMPLE 2 Toxicity Study of the Anti-mouse Fas Ligand Antibody FLIM58

(1) Method

Male, 8 week old DBA/1J mice and C3H/He mice (Charles River Japan ) wereused. The mice were administered i.v. via their tail vein with theanti-mouse Fas ligand antibody FLIM58 at a dose of 100 mg/30 ml/kg. Thecontrol group was administered i.v. via their tail vein withphysiological saline at a dose of 30 ml/kg. Each groups consisted ofthree animals for both strains. Observation period was/days, and bodyweight measurement, hematological tests (red blood cell, white bloodcell, platelet), and hematobiological tests (GOT, GPT, urea nitrogen),and autopsy with naked eye were conducted.

(2) Results

The body weight increase, the hematological test values (red blood cell,white blood cell, platelet), and the hematobiological test values (GOT,GPT, urea nitrogen) of the group administered with the anti-mouse Fasligand antibody FLIM58 were not significantly different from those ofthe control group. In addition, no abnormalities were observed in thegroup administered with the anti-mouse Fas ligand antibody FLIM58 by theautopsy with naked eye.

EXAMPLE 3 Effect of Anti-mouse Fas Ligand Antibody FLIM58 in Improvingthe Pathology of Rat EAE Model (Adoptive Transfer Model)

(1) Preparation of Rat Adoptive Transfer Model

10 ml of Freund's complete adjuvant containing 1 mg/ml of killedMycobacterium tuberculosis H37Ra (manufactured by Difco Laboratories)was centrifuged at 1000 rpm for 5 minutes, and the resulting precipitatewas again suspended in 1.6 ml of Freund's incomplete adjuvant(manufactured by Difco Laboratories) to prepare a complete adjuvant withhigher concentration of H37Ra. Myelin basic protein from guinea pigbrain (manufactured by Sigma) was dissolved in physiological saline to2.5 mg/ml, and the solution was mixed at 1:1 with the complete adjuvantof higher killed Mycobacterium tuberculosis H37Ra concentration asdescribed above. The mixture was emulsified using. Leur lock Hamiltongastight syringe (manufactured by Chuo-Kagaku-Kogyo) to prepare anemulsion.

A female, 11 week old Lewis rat was anesthetized by intraabdominallyadministering 50 mg/kg of pentobarbital (manufactured by DainihonPharmaceuticals), and the rat was injected into both of its hind legs(foot pads) with 0.1 ml of the emulsion as described above,respectively. At day 14 after the injection, spleen was extirpated, andthe spleen was disintegrated with forceps, centrifuged, and theresulting cell precipitate was suspended in 0.017M Tris-0.747% ammoniumchloride solution for selectively lying the erythrocytes. The remainingcells were washed in Hanks's solution (manufactured by NissuiPharmaceuticals) to obtain the splenocytes. The splenocytes wereinoculated at a concentration of 4×10⁶ cells/ml in RPMI1640 medium(manufactured by GIBCO BRL) supplemented with 25 μg/ml myelin basicprotein from guinea pig brain, 2 mM L-glutamine (manufactured by NissuiPharmaceuticals), and 10% inactivated FBS (manufactured by JRHBioscience), and cultured at 37° C. in the presence of 5% carbon dioxidegas for 3 days. The culture medium of the splenocytes was centrifuged at1,000 rpm for 5 minutes, and the precipitated cells were again suspendedin Hanks's solution. The splenocytes were transplanted into theabdominal cavity of female, 11 week old Lewis rat at a dose of 1.2×10⁷cells/2 ml/rat to prepare the EAE model.

(2) Administration of Anti-mouse Fas Ligand Antibody FLIM58

The rats were administered with 10 mg/kg of anti-mouse Fas ligandantibody FLIM58 at the day of splenocyte transplantation (day 0), andafter 6 days from the splenocyte transplantation(day 6) i.v. via theirtail vein. The control group was administered with equal dose of IgGpurified from normal hamster γ-globulin (manufactured by ROCKLAND) usingprotein A column. Each group consisted of 5 rats.

(3) Evaluation

The effects of FLIM58 administration was evaluated by scoring thepathology in accordance with the criteria shown in Table 1, below(Experimental Neurology, vol. 151, pages 221-228, 1995).

TABLE 1 Pathological Score of Rat EAE model Score Pathology Score 0Normal Score 0.5 Incomplete paralysis in tail (The tail raised dropsdown at a timing earlier than the normal rat) Score 1 Complete paralysisin tail (The tail raised quickly drops down) Score 2 Paralysis in one ofthe hind legs Score 3 Paralysis in both of the hind legs Score 4 DyingScore 5 Dead

(4) Results

The results are shown in FIGS. 1 and 2. The pathological conditions wereobserved at day 5 to day 6 (5 to 6 days after transplantation,hereinafter the same abbreviations are used), and after day 6, theconditions of the FLIM58 administrated group were less severe comparedto the control group. The loss of body weight associated with onset ofthe pathology started from day 4 to day 5, and after day 5, the weightloss of the FLIM58 administrated group was less than that of the controlgroup.

EXAMPLE 4 Effect of Anti-mouse Fag Ligand Antibody FLIM58 in Improvingthe Pathology of Rat EAE Model (Active Immunization Model)

(1) Preparation of Rat Actively Immunization Model

The procedure of Example 3 was repeated to produce the 1:1 emulsion ofmyelin basic protein and Freund's complete adjuvant. The emulsion wasadministered to both foot pads of the rat at a dose of 0.1 ml/foot pad(0.2 ml/rat) under anesthetization.

(2) Administration of Anti-mouse Fas Ligand Antibody FLIM58

The rats were administered with 10 mg/kg of anti-mouse Fas ligandantibody FLIM58 7 days after myelin basic protein (day 7) i.v. via theirtail vein. The control group was administered with equal dose of IgGpurified from normal hamster γ-globulin. Each group consisted of 5 rats.

(3) Evaluation

The effects of FLIM58 administration was evaluated by scoring thepathology in accordance with the criteria shown in Table 1.

(4) Results

The pathological conditions started at day 10, and the conditions of theFLIM58 administrated group were less severe compared to the controlgroup.

Industrial Applicability

The preventive and therapeutic agent for autoimmune demyelinatingdiseases of the present invention contains an apoptosis-suppressingsubstance a its effective component, and it has the action ofsuppressing the apoptosis. Therefore, the agent of the present inventionhas the effects of preventing or treating the autoimmune demyelinatingdiseases wherein apoptosis such as the biological actions related withFas/Fas ligand system is involved. Exemplify such biological actions isFas-mediated cell death. The apoptosis-suppressing substance of thepresent invention is highly expected for use as a prophylactic andtherapeutic agent for the autoimmune demyelinating diseases whereinapoptosis such as the Fas-mediated cell death is involved.

What is claimed is:
 1. A method for treating autoimmune demyelinatingdiseases which comprises administering to a patient in need thereof aneffective amount of a Fas antagonist, which is a substance that binds toFas ligand and inhibits Fas-Fas ligand binding and suppresses apoptosis,in myelin sheath cells.
 2. The method according to claim 1 wherein saidFas antagonist is an anti-Fas ligand antibody.
 3. The method accordingto claim 1 wherein said autoimmune demyelinating disease is a diseaseassociated with demyelination in central nervous system.
 4. The methodaccording to claim 1 wherein said autoimmune demyelinating disease is atleast one member selected from acute disseminated encephalomyelitis andmultiple sclerosis.
 5. A method of treating a disease associated withdemylination in the central nervous system which comprises administeringto a patient in need thereof, an effective amount of an anti-Fas ligandantibody.
 6. A method of treating multiple sclerosis which comprisesadministering to a patient in need thereof an effective amount of ananti-Fas ligand antibody.
 7. A method of treating a disease associatedwith demylination in the central nervous system which comprisessuppressing apoptosis with an effective amount of an anti-Fas ligandantibody.
 8. A method of treating multiple sclerosis which comprisessuppressing apoptosis with an effective amount of an anti-Fas ligandantibody.